The information disclosed in this Specification was made in part with Government grant support, awarded by the National Institute of Health. The government may have certain rights in the invention disclosed in this Specification.
This invention relates generally to the field of N-proteinase and the production, uses and methods thereof.
The ExtraCellular Matrix. The most abundant component of the extracellular matrix is collagen. Collagen molecules are generally the result of the trimeric assembly of three polypeptide chains containing, in their primary sequence, (-Gly-X-Y-)n repeats which allow for the formation of triple helical domains. Van der Rest et al. 1991, FASEB J. 5:2814-2823.
During their biosynthesis, the fibrillar collagens, including collagen types I, II and III, are synthesized as precursors, known as procollagens. These procollagens are comprised of a central triple-helical collagen domain extended by propeptides both at the molecules"" carboxyl and amino ends. These propeptides, designated as C-propeptide (for the propeptide found at the carboxyl terminal end of procollagen) and N-propeptide (for the propeptide found at the amino terminal end of procollagen), are cleaved during post-translational events by the enzymes C-proteinase and N-proteinase, respectively.
Diseases Associated with the Abnormal Production of Collagen. An array of critical diseases has been associated with the inappropriate or unregulated production of collagen, including pathological fibrosis or scarring, including endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers"" fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, binary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, scleroderma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne""s), cardiac fibrosis, muscular fibrosis/retinal separation, esophageal stricture, payronles disease. Further fibrotic disorders may be induced or initiated by surgery, including scar revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren""s contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis. One strategy for the treatment of these diseases is the inhibition of the pathological overproduction of collagen. The identification and isolation of enzymes involved in the collagen production and processing are therefore of major medical interest to provide for suitable targets for drug development.
Similarly, a strategy for the treatment of diseases resulting from the pathological underproduction of collagen, where the underproduction of collagen is the consequence of improper processing of procollagen, is the administration of C-proteinase.
N-Proteinase. N-proteinase is the post-translational enzyme responsible for cleaving the N-propeptide from the procollagen molecule. Type III N-proteinase is specific to type III procollagen and excises the N-propeptide from type III procollagen only. In contrast, type I N-proteinase acts on both collagen types I and II.
The purification of both type I and type III N-proteinase from natural sources, including chicken embryos, has been previously reported. For example, with respect to type III N-proteinase, the isolation and purification of human enzyme from ascitic fluid and placenta were reported in 1985 and 1986, respectively. See, Niemela et al., 1985, Biochem. J. 232:145-150; Halila and Peltonen, 1986, Biochem. J. 239:47-52. The isolation and at least partial purification of type I N-proteinase from chick and bovine sources, have also been reported previously. See, Kohn et al., 1974, Proc. Natl. Acad. Sci. USA 71:44; Tuderman and Prockop, 1982, Eur. J. Biochem. 125:545-549; Tazawa et al., 1985, J. Biol. Chem. 260:1120-1126; Hojima et al., 1994, J. Biol. Chem. 269:11381-11390; Colige et al., 1995, J. Biol. Chem. 270:16724-16730.
The kinetics of purified naturally-occurring N-proteinases, both Types I and III, have also been studied. Dombrowski and Prockop, 1988, J. Biol. Chem. 263:16545-16552. Prior to the present invention, however, the nucleotide sequence of N-proteinase had not been determined and thus the means for producing recombinant N-proteinase was unknown.
N-proteinase exists in two forms, a xe2x80x9cshortxe2x80x9d form comprising a molecule approximately 70 kDa in length and a xe2x80x9clongxe2x80x9d form comprising a molecule approximately 130 kDa in length. The present invention is directed to polynucleotide sequences encoding both the short and long forms of N-proteinase, including fragments of both forms of N-proteinase having the ability to cleave N-propeptide from procollagen.
The present invention is further directed to synthesized or recombinant compositions corresponding to or derived from the polynucleotide sequences of the present invention. In one embodiment of the present invention, the composition is radiolabelled for use in assays.
The present invention is also related to the synthesis of recombinant production of N-proteinase and related compositions. Where N-proteinase is produced recombinantly, the use of a variety of recombinant expressions systems is contemplated, including yeast, plant cell, insect cell, mammalian cell and E. coli expression systems.
As used in this Specification, the term xe2x80x9cN-Proteinasexe2x80x9d shall mean: (1) a protein encoded by the amino acid sequence as set forth at FIG. 1D (SEQ ID NO:5) deduced from the nucleic acid sequences set forth at FIGS. 1A-1C, (SEQ ID NO:1) a protein encoded by the amino acid sequences as set forth at FIG. 2B (SEQ ID NO:7) deduced from the nucleic acid sequence set forth at FIG. 2A, (SEQ ID NO:6) and the amino acid sequence encoded from the nucleic acid sequences set fort at FIGS. 4A-4B; (SEQ ID NO:8) (2) a protein having N-proteinase activity wherein such protein is encoded by the amino acid sequences deduced from the nucleic acid sequences set forth at FIGS. 1A-1C, (SEQ ID NO:1) FIG. 2A, (SEQ ID NO:6) and FIGS. 4A-4B, (SEQ ID NO:8) wherein one or more amino acids have been added, deleted, mutated, substituted or otherwise altered (xe2x80x9cderivativexe2x80x9d) and the nucleotide sequence encoding said protein can hybridize to the nucleic acid sequence of FIGS. 1A-1C, (SEQ ID NO:1) FIG. 2A (SEQ ID NO:6) and FIGS. 4A-4B (SEQ ID NO:8) under stringent hybridization conditions; (3) a fragment of N-proteinase or a derivative thereof; and (4) the protein encoded by a naturally-occurring allele or homolog of the gene corresponding to the nucleic acid sequences set forth at FIGS. 1A-1C, (SEQ ID NO:1) FIG. 2A, (SEQ ID NO:6) or FIGS. 4A-4B (SEQ ID NO:8).
As used in this Specification, the term xe2x80x9cPolynucleotidexe2x80x9d denotes DNA, cDNA and/or RNA, including genomic DNA and mRNA.
As used in this Specification, the phrase xe2x80x9cStringent Hybridization Conditionsxe2x80x9d refers to those hybridizing conditions that (1) employ low ionic strength and high temperature for washing, for example, 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50xc2x0 C.; (2) employ during hybridization a denaturing agent such as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42xc2x0 C.; or (3) employ 50% formamide, 5xc3x97SSC (0.75 M NaCl, 0.075 M Sodium pyrophosphate, 5xc3x97Denhardt""s solution, sonicated salmon sperm DNA (50 xcexcg/ml), 0.1% SDS, and 10% dextran sulfate at 42xc2x0 C., with washes at 42xc2x0 C. in 0.2 xc3x97SSC and 0.1% SDS.
As used in this Specification, the phrase xe2x80x9cRecombinant Expression Vectorxe2x80x9d refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the N-proteinase sequences.